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Development of a quantification method of HcRNAV infectious bloom-forming dinoflagellate Heterocapsa circularisquama with a monoclonal antibody and PCR

Research Project

Project/Area Number 24580290
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field General fisheries
Research InstitutionFisheries Research Agency

Principal Investigator

NAKAYAMA Natsuko  独立行政法人水産総合研究センター, 瀬戸内海区水産研究所, 研究員 (20612675)

Co-Investigator(Kenkyū-buntansha) NAGASAKI Keizo  独立行政法人水産総合研究センター, 本部, 研究開発コーディネーター (00222175)
HAMAGUCHI Masami  独立行政法人水産総合研究センター, 瀬戸内海区水産研究所, 主幹研究員 (60371960)
Project Period (FY) 2012-04-01 – 2015-03-31
Project Status Completed (Fiscal Year 2014)
Budget Amount *help
¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2013: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2012: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
KeywordsmRT-PCR / HcRNAV / ELISA / ヘテロカプサ / リアルタイムPCR / Real-time PCR / H. circularisquama / Quantification
Outline of Final Research Achievements

We established the Enzyme-linked Immunoadosorbent Assay (ELISA) system and the multiplex real-time reverse transcription PCR (mRT-PCR) system for quantifying a single-stranded RNA virus (HcRNAV) that causes a lytic infection in bloom-forming dinoflagellate Heterocapsa circularisquama, and checked its availability to environmental samples. In mRT-PCR, two primer-probe sets targeting separate conserved regions in the HcRNAV genome allowed highly specific detection of HcRNAV. The accuracy of the system was tested using three typical HcRNAV strains by comparing the enumeration results of the mRT-PCR method and two conventional methods, transmission electron microscopy (TEM) and a most-probable-number (MPN) assay. As a result, estimates obtained via the mRT-PCR method were consistent with those of TEM, indicating that it is a useful method for quantifying HcRNAV. In the ELISA, a monoclonal and polyclonal antibody established became possible to specifically detect the HcRNAV in situ.

Report

(4 results)
  • 2014 Annual Research Report   Final Research Report ( PDF )
  • 2013 Research-status Report
  • 2012 Research-status Report
  • Research Products

    (4 results)

All 2014 2013

All Presentation (4 results)

  • [Presentation] Prospect for the biological control of Heterocapsa circularisquama bloom by inoculating frozen bottom sediment with HcRNAV viruses.2014

    • Author(s)
      Natsuko Nakayama, Shinichi Kondo, Naotsugu Hata, Yuji Tomaru, Masami Hamaguchi, Keizo Nagasaki and Shigeru Itakura
    • Organizer
      PICES2015
    • Place of Presentation
      韓国
    • Year and Date
      2014-10-17
    • Related Report
      2014 Annual Research Report
  • [Presentation] マルチプレックスリアルタイムRT-PCRを用いた海底泥からのウイルスHcRNAV定量の検証2014

    • Author(s)
      中山奈津子・浜口昌巳・畑直亜・渡邊和博・長崎慶三
    • Organizer
      平成26年度日本水産学会春季大会
    • Place of Presentation
      北海道大学函館キャンパス
    • Related Report
      2013 Research-status Report
  • [Presentation] Enumeration of a Dinoflagellate-infecting ssRNA virus by Multiplex real-time PCR.2013

    • Author(s)
      N. Nakayama, K. Nagasaki, N. Hata, K. Watanabe, M. Hamaguchi
    • Organizer
      7th Aquatic Virus Workshop
    • Place of Presentation
      US フロリダ
    • Related Report
      2013 Research-status Report
  • [Presentation] 有害渦鞭毛藻感染性ウイルスHcRNAVに対するマルチプレックスリアルタイムRT-PCR法の開発2013

    • Author(s)
      中山奈津子, 長崎慶三, 浜口昌巳
    • Organizer
      第29回日本微生物生態学会
    • Place of Presentation
      鹿児島大学
    • Related Report
      2013 Research-status Report

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Published: 2013-05-31   Modified: 2019-07-29  

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